1. Field of Invention
The present invention relates generally to methods and systems for detecting a microorganism, and more particularly, to methods and systems for lysing of the cellular membrane of the microorganism and using highly sensitive microwave-accelerated metal-enhanced fluorescence (MAMEF) technology.
2. Description of the Related Art
The ability to accurately identify biological organisms and/or biological pathogens that threaten the health of humans in real time will enable clinicians to make informed decisions about the most appropriate countermeasures.
Salmonella, a genus of more than 2500 serological variants (serovars), includes many organisms that can cause human disease. Salmonella enterica subsp. enterica Typhi and S. Paratyphi A and B cause, respectively, typhoid and paratyphoid fevers (enteric fevers), febrile illnesses characterized by infection of the gut-associated lymphoid tissue, liver, spleen, bone marrow and gall bladder and accompanied by a low level bacterium.[1] Non-typhoidal Salmonella (NTS) generally produce a self-limited gastroenteritis (vomiting, fever and diarrhea) in healthy hosts.[2-4] By contrast, in young infants, the elderly and immunocompromised hosts, NTS can cause severe, fatal disease in the USA [4,5] and abroad.[2, 6-12]
The most common serotypes isolated from blood in the USA are S. Typhimurium (24%), S. Enteritidis (19%) and S. Heidelberg (15%).[13] S. Typhimurium and S. Enteritidis are also the most commonly isolated NTS serovars from blood and other normally sterile sites in Europe,[14,15] United Kingdom[16] and in Africa.[9,10,17-19] S. Typhimurium and S. Enteritidis account for 80-90% of all invasive NTS in sub-Saharan Africa.[6, 9, 10, 12, 17-21]
Invasive Salmonella spp. is routinely detected by standard blood culture techniques. Culturing blood specimens has become much faster and easier since the advent of continuously monitoring blood culture instruments such as the BACTEC 9000 systems (Becton Dickinson, Cockeysville, Md.). However, it still takes several days for detection of Salmonella. For example, using the BACTEC 9240 system, of 21 Salmonella spp. recovered from blood culture bottles, 13 were detected on day 1, 7 on day 2 and 1 on day 6.[22] Another study using the BACTEC 9120 found that the 36 Salmonella spp. isolated were detected between 14.51 and 31.96 hours (median detection time of 19.48 hours).[23]
Following detection, the bacteria must still be isolated from the blood and identified by standard microbiological techniques and the serovar is ascertained by agglutination with commercial antisera (normally at a reference laboratory). Due to the time required for blood culture identification and the fact that many diagnostic labs are unable to serotype Salmonella spp. themselves, alternative methods of identification of Salmonella are being sought.[24] In particular, DNA detection tests such as PCR have been investigated. The food industry routinely uses PCR to detect Salmonella in food. [25, 26]
There are many reports of PCR primers designed to detect S. Typhi from the blood of enteric fever patients.[27-35] Furthermore, the sensitivity of PCR has often been found to be higher than that of blood culture.[29,30,32,33] However, PCR has not yet become an established method for diagnosis of typhoid fever.[24] One reason for this may be that although some reports claim high sensitivity, with detection of as few as 10 CFU/mL of blood,[34] Wain et al.'s prospective study of the concentration of S. Typhi in blood of typhoid fever patients showed a median value of 0.3 (range of 0.1 to 399) CFU/mL, well below current PCR-based detection limits.[36] Interestingly, in another study, Wain et al. [37] showed that 63% of the S. Typhi cells were located in the buffy coat layer (presumably in monocytes and polymorphonuclear leukocytes) and the mean number of bacteria per infected leukocyte was 1.3 CFU/cell. The quantitative cultures of Wain et al. corroborate the classic early study of Watson [38] who showed a median of 6 CFU/ml of S. Typhi in 15 patients with typhoid fever. NTS have been shown to be present at a similar concentration (M. A. Gordon, unpublished results). Detection of NTS directly from blood has not been investigated.
Another bacteria pathogen that requires a point of care testing method is Chlamydia trachomatis (CT) which is the most prevalent bacterial sexually transmitted infection (STIs) reported to the Centers for Disease Control and Prevention (CDC)[56]. There were 1.2 million cases of Chlamydia reported to the CDC in 2008. CDC estimates that STIs cost the health care system $1.5 billion annually [57]. Since these infections are most often asymptomatic, the CDC and other professional organizations recommend yearly screening for Chlamydia in all sexually active women ages 16-25 years of age[58]. Although there are several commercial assays available for performing nucleic acid amplifications tests (NAATs) [59-61], which are now recommended by CDC as the test of choice (APHL), they are time consuming, not convenient for use and not considered point of care tests. Currently there are no commercially available POC assays for the detection of Chlamydia that have rapid, high enough sensitivity and specificity to be recommended [62]. In this regard, there is a great need to develop rapid detection technologies for testing Chlamydia infections using point of care (POC) assays in order to expedite immediate diagnosis and treatment in both private and public health care settings.
The need for “real-time” (<60 mins) detection has lead to the development of technologies based on DNA (PCR) and protein (antibody) targets. PCR and reverse transcriptase PCR assays have been reported for detecting the pathogen anthrax in air samples. However, these advances are not considered simple or monetarily reasonable, and therefore, limit their potential as field-deployable, emerging technologies for use in ultra-sensitive pathogen detection. Thus, there is a pressing need for a sensitive and specific rapid diagnostic test to detect pathogens and preferably able to differentiate between multiple pathogenic agents, that does not suffer from the problems of the prior art and does not require any amplification steps, such as in PCR or ELISA.